Project Summary The early C. elegans embryo provides a powerful model system for molecular genetic studies of cell division. During the current funding period, (i) we have obtained evidence that receptor-independent G[unreadable] proteins regulate non-muscle myosin II to influence positioning of the first mitotic spindle in one-cell zygotes. (ii) We have isolated a large collection of mutants with mitotic spindle defects led to our discovery that the spindle assembly checkpoint is active in early C. elegans embryos, and to our identification of several genes required for spindle assembly or function. (iii) We developed a powerful genome-wide RNA interference-based modifier screen that has identified conserved genes not previously known to influence mitotic spindle assembly and function. In addition, through our use of this modifier screen we have found that dynein light chains can negatively regulate dynein heavy chain function, which to our knowledge has not been documented previously. In this renewal, we propose (i) to further study a group of 21 specific suppressors of conditional dynein heavy chain mutants, and test our hypothesis that dynein light chains negatively regulate dynein function in early embryonic cells. We also will (ii) extend our use of novel particle tracking methods, and other approaches, to further test our hypothesis that a network of cortical and cytoplasmic actomyosin is regulated by G[unreadable] proteins and applies forces to astral microtubules that contribute to mitotic spindle positioning. In a final aim (iii), we will characterize and positionally clone the genes mutated in new conditional mutants we have identified in five different loci that are required for mitotic spindle assembly, positioning, or function. We also will pursue further analysis of several specific suppressors of two conditional mutants with defects in centrosome maturation, to extend our identification and analysis of conserved genes that influence mitotic spindle assembly and function. These studies of cell division and cytoskeletal function are relevant to several important human diseases. Advances in our understanding of these processes, and in the identification of conserved genes that influence them, may prove useful in developing therapeutic interventions for the treatment of human disease. Project Narrative The essential cell division processes we study, using the early Caenorhabditis elegans embryo as a model system, are highly conserved in all animals, including humans, and are relevant not only to cancer, but also to many other human illnesses, including neurodegenerative diseases. Chemotherapeutic agents such as taxol, which target the cell division machinery, can be useful for inhibiting cancer cell growth: a better understanding of the machinery, including further identification of the parts, will provide more candidate targets for therapeutic intervention. Lastly, our studies of the asymmetric cell divisions that characterize early embryogenesis in C. elegans also are relevant to our understanding of stem cell biology, and our ability to manipulate stem cell development for therapeutic applications.